The invention relates to the identification and cloning of coding sequences for proteases, particularly novel proteases.
Proteases are involved in a variety of cellular processes, such as tumor invasion, wound healing, tissue remodeling, infection, and inflammation. Previously unknown proteases whose substrate specificities are known can be used, for example, to design compounds which can affect these processes, including therapeutic compounds for conditions such as cancer, inflammation, rheumatoid arthritis and other autoimmune diseases, and AIDS.
Many methods are available in the art for detecting protease activity. For example, WO 97/08194 allegedly discloses a method of assaying for protease activity by measuring the fluorescence intensity of a fluorescent substrate. It would be advantageous to have a method in which detection of protease activity is linked to identification of a polynucleotide encoding the protease. Thus, there is a need in the art for convenient and rapid methods which can be used both for detection of protease activity and for identification of polynucleotides encoding the protease.
It is an object of the present invention to provide methods for identifying coding sequences for proteases which are expressed under a variety of conditions, including proteases which were previously unknown. This and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention is a method of identifying a candidate polynucleotide molecule encoding a protease, such as a cell surface or a secreted protease. Viral display packages are contacted with target cells. The viral display packages comprise a transferable label and display a chimeric envelope protein comprising (I) a substantially intact viral envelope protein which enhances fusion between a viral display package and a target cell membrane, (ii) an inhibitory protein which prevents fusion between the viral display package and the target cell membrane, and (iii) a protease recognition site located between the substantially intact viral envelope protein and the inhibitory protein. In another embodiment, the viral display packages display recombinant envelope proteins, in which a protease recognition site has been substituted for a grin cleavage site located between a large glycoprotein subunit of the envelope protein and a transmembrane component of the envelope protein. Recombinant envelope proteins can be derived from viral envelope proteins such as a Moloney murine leukemia virus envelope protein or an influenza virus envelope protein.
A xe2x80x9cprotease recognition sitexe2x80x9d according to the invention is a contiguous sequence of amino acids connected by peptide bonds which are recognized by a protease. Recognition of this site by a protease results in cleavage (i.e., hydrolysis) of peptide bond by the protease. The site of hydrolysis may be coincident with the protease recognition site, that is, the protease recognition site may include one or more amino acids on either side of the peptide bond to be hydrolyzed which are recognized by the particular protease. The specific sequence of amino acids in the protease recognition site depends on the catalytic mechanism of the protease, which is defined by the nature of the functional group at the protease""s active site. Alternatively, the protease recognition site may be one, two, three, four or more amino acids distal, at the amino or carboxy terminus, to the site of cleavage by the protease. If desired, the protease recognition site can be a variation of a recognition site of a known protease. Preferably, the variation of the known recognition site is formed by modifying at least one amino acid of the known protease recognition site.
Preferably, the substantially intact retroviral envelope protein is a murine leukemia virus envelope protein, such as a 4070A or Moloney murine leukemia virus envelope protein.
In one embodiment, the inhibitory protein binds to a receptor present on the outer cell membrane of the target cell. The inhibitory protein can be, for example, CD3 antigen, epidermal growth factor, stem cell factor, and an insulin-like growth factor I. In another embodiment, a first inhibitory protein oligomerizes with at least second inhibitory protein, such as CD40 ligand or a leucine zipper polypeptide, for example GCN4, C/EBP, Fos, Jun, and c-myc.
A transferable label can be any label whose presence can be detected in the target cell upon fusion of the viral display package and the target cell membrane Preferably, the transferable label is a gene encoding a selectable marker or is a reporter gene.
Target cells comprise(I) expressible candidate polynucleotide molecules, wherein the expressible candidate polynucleotide molecules may or may not encode a protease. The target cell can be a cell which is likely to express a protease, such as a tumor cell, a cell of a tissue which is inflamed, a cell of a tissue which is undergoing remodeling, a cell of a tissue which is involved in wound healing, or a cell comprising an infectious agent which expresses a protease. If desired, candidate polynucleotide molecules can be obtained from such cells and introduced into target cells. Candidate polynucleotides also can be obtained from cells which are known to express a protease or which are not known to express a protease. Optionally, candidate polynucleotides are synthetic polynucleotides or are polynucleotides which encode a mutated form of a known protease. Candidate polynucleotides can be obtained from a cDNA library.
Infection of a target cell by a viral display package comprising the transferable label preferably occurs only if a protease produced in the target cell removes the inhibitory protein from the chimeric envelope protein or, if a recombinant envelope protein is displayed, only if a protease produced in a member of the first plurality of target cells activates the envelope protein by cleaving it at the protease recognition site between the large glycoprotein subunit and the transmembrane component. Target cells may be treated with growth factors, activating proteases or other compounds to modulate the protease activity of the target cells,
As used herein, the term xe2x80x9ctreatment to modulate protease activityxe2x80x9d refers to a process or treatment that results in activation of a protease expressed in a zymogen or pro-enzyme form. This treatment may, for example, activate or even introduce a proteolytic enzyme required for cleavage of a zymogen form of a protease.
As used herein, the term xe2x80x9cgrowth factorxe2x80x9d refers to a polypeptide that alters protease activity in a cell through interaction of the polypeptide with a specific receptor expressed by that cell.
As used herein, the term xe2x80x9cactivating proteasexe2x80x9d refers to a proteolytic activity that, through cleavage of one or more polypeptides, modulates the activity of a cellular protease.
As used herein, the term xe2x80x9cprotease-modulating compoundxe2x80x9d refers to a compound that directly or indirectly activates a cellular protease. Detection of a target cell which comprises the transferable label thus indicates that the target cell expresses the protease.
Primers can be included in the candidate polynucleotide molecules, for use in amplifying a candidate polynucleotide molecule which encodes a protease. Amplified candidate polynucleotide molecules can then be sequenced to identify the encoded protease.
Thus, the present invention provides an innovative approach to the identification of protease coding sequences, including those which are novel or have altered substrate specificities.